Project Summary/Abstract While many see carbon monoxide (CO) as strictly a toxic gas, CO is also produced in the body from the natural breakdown of heme by heme oxygenase enzymes (HO-1 and HO-2). Recent laboratory studies have shown that exposure to moderate levels of CO will elicit potent cytoprotective effects against hypoxic and ischemic events. These properties have led to the investigation of the therapeutic potential of CO. However, the optimal CO levels through which the safest and the most potent therapeutic effect can be achieved is still unknown. Model systems which exhibit increased endogenous CO as a protective strategy, rather than pathological side effect, will provide insight into CO exposure levels that are safe and effective. Our preliminary work has revealed two human populations and one diving mammal, all adapted to chronic hypoxia, that express increased CO production or positive selection involving the HO-2 gene. Our previous work with Tibetan genomes revealed positive selection at the HO-2 locus, suggesting an important role of the HO/CO pathway in high-altitude adaptation. Similarly, our preliminary end-tidal CO measurements in Peruvian natives show that high-altitude natives have increased end-tidal CO compared to low altitude natives. Likewise, my dissertation work has shown that elephant seals are the only mammal known to produce and maintain CO at the same moderate levels recently deemed therapeutic and protective in the human and laboratory animal studies mentioned above. Elephant seals exhibit repeated, voluntary sleep apnea events (~ 10-15 min) when on land, where they are known to regularly experience degrees of hypoxia and tissue ischemia which would elicit detrimental effects in other mammals. Due to this preliminary evidence, I propose that high-altitude natives and elephant seals represent ideal models to improve our understanding on the mechanisms behind the natural upregulation of the HO/CO pathway in alleviating hypoxia-induced injuries. Specifically, this proposal outlines a multidisciplinary approach into the investigation of the cellular and genetic mechanisms behind the natural upregulation of the HO/CO pathway, and explores the associated tissue-specific protective properties. Humans and elephant seals will be sampled during periods of chronic hypoxia and normoxia. The quantity and activity of HO-1, HO-2 and biliverdin reductase (BVR) will be evaluated in the blood (plus skeletal muscle in elephant seals) from all patients. The precursors (hemoglobin and heme) and products (i.e. CO, iron, biliverdin and bilirubin) of HO and BVR activity will be measured in the same blood samples. The removal rates of CO will be determined through end-tidal CO values and the excretion of bilirubin breakdown products (stercobilin and urobilin) will be measured in the feces and urine. The heme store removal will be measured by investigating red blood cell lifespan. To evaluate the genetic regulation of this pathway, transcriptomics on RNA from the blood samples (plus skeletal and liver tissue in elephant seals) taken after hypoxic and normoxic periods will demonstrate the upregulation or downregulation of specific genes in relation to the HO/CO pathway activity and oxygen availability in the two states. Markers of anti-inflammation, anti- apoptosis, anti-proliferation, and anti-oxidation will be measured in blood and tissue samples and will be compared between groups and to gene expression values and the activity of the HO/CO pathway.